A real-time bioluminescent HTS method for measuring protein kinase activity influenced neither by ATP concentration nor by luciferase inhibition.

The firefly luciferin-luciferase reaction has been used to set up an assay for protein kinase based on measuring ATP consumption rate as the first-order rate constant for the kinase reaction. The assay obviates the problems encountered with previous bioluminescent protein kinase assays such as interference with the luciferase reaction from library compounds, nonlinear standard curves, and limited dynamic ranges. In the assay described in the present paper luciferase and luciferin are present during the entire kinase reaction, and the light emission can be measured continuously. In an HTS situation the light emission is measured only twice, i.e., initially and after a predetermined time. After a fivefold reduction of the ATP concentration a Z' value of 0.96 was obtained. Light emission data from samples with kinase are normalized with light emission data from blanks without kinase. First-order rate constants for the kinase reaction calculated from normalized light emission are not affected by a moderate degree of inactivation of luciferase and luciferin during the measuring time. The constants have the same value at all ATP concentrations much lower than the K(m) of the luciferase and the kinase. These factors make the assay very robust and influenced neither by ATP concentration nor by luciferase inhibition. The measuring time depends on the kinase activity and can be varied from minutes to more than 8 h provided the kinase is stable and the evaporation of water from the wells is acceptable. The assay is linear with respect to kinase activity over three orders of magnitude. The new reagents also allowed us to determine K(m) values for ATP and for Kemptide.